Rigidized trimethine cyanine dyes

ABSTRACT

Disclosed are analogues of trimethine cyanine dyes, which are useful for importing fluorescent properties to target materials by covalent and non-covalent association.

The present application is a divisional of U.S. application Ser. No.10/351,369, filed Jan. 27, 2003 (allowed), which is a continuation ofU.S. application Ser. No. 09/639,941, filed Aug. 17, 2000 (abandoned),which is a continuation of U.S. application Ser. No. 09/212,564, filedDec. 16, 1998 (now U.S. Pat. No. 6,133,445); Ser. No. 09/639,941 is acontinuation of PCT/US98/26665, filed 16 Dec. 1998, which designated theU.S. and is a continuation-in-part of U.S. application Ser. No.08/992,212, filed Dec. 17, 1997 (abandoned), and Ser. No. 09/212,564 isa CIP of Ser. No. 08/992,212, the entire contents of each of which ishereby incorporated by reference in this application.

The present invention relates to rigidized trimethine cyanine dyes,their preparation, their use as fluorescent markers and in fluorescenceenergy transfer complexes and to materials labelled with them.

Fluorescent dyes are generally known and used for fluorescence labellingand detection of various biological and non-biological materials byprocedures such as fluorescence microscopy, fluorescence immunoassay andflow cytometry. A typical method for labelling such materials withfluorescent dyes is to create a fluorescent complex by means of bondingbetween suitable groups on the dye molecule and compatible groups on thematerial to be labelled. In this way, materials such as cells, tissues,amino acids, proteins, antibodies, drugs, hormones, nucleotides, nucleicacids, lipids and polysaccharides and the like may be chemicallylabelled and detected or quantitated, or may be used as fluorescentprobes which can bind specifically to target materials and detected byfluorescence detection methods.

Four commonly used classes of fluorescent dyes are those based on thefluorescein (green fluorescence), rhodamine (orange fluorescence),coumarin and pyrene (blue fluorescence) chromophores. Dyes based onfluorescein and rhodamine have a number of disadvantages. Fluoresceinderivatives have a pH-sensitive absorption spectrum and fluorescenceyield decreases markedly below pH 8. Rhodamine derivatives arehydrophobic and are difficult to use in aqueous media. They often showstrong fluorescence quenching when bound to proteins.

U.S. Pat. No. 5,268,486 discloses luminescent mono- and polymethinecyanine dyes and related polymethine dyes such as merocyanine and styryldyes which contain groups enabling them to covalently attached to amine,hydroxyl, aldehyde and sulphydryl groups on a target material. Thecompounds are disclosed as fluorescing in the green, orange, red andnear infra-red regions of the spectrum.

U.S. Pat. No. 3,679,427 describes rigidized cyanine dyes which contain atrimethine chain as part of a rigid structure, as shown in formula (1):

where each of Z and Z¹ represents the non-metallic atoms necessary tocomplete a heterocyclic nucleus of the type used in cyanine dyes; Rrepresents a member selected from a hydrogen atom, an alkyl radical, oran aryl radical; R¹ represents a member selected from oxygen, sulphur,selenium or nitrogen. The subject dyes are reported to exhibit strongfluorescence and are useful spectral sensitizing dyes for photographicsilver halide as well as being useful as colorant materials for a widevariety of compositions such as paints, lacquers, etc. However they arenot described as fluorescent labelling dyes.

European Patent Application No. 747448 describes bis-heterocyclicmonomethine cyanine dyes, rigidized by means of a bridging group betweenthe nitrogen atoms of the heterocycles. Such compounds may besubstituted with additional groups chosen to provide desirablesolubility, reactivity and spectroscopic properties to the fluorescentcompounds. The dyes can be used to covalently label a target material soas to impart fluorescent properties to that target. The monomethinerigidized cyanines are highly fluorescent and strongly light-absorbingdyes which emit in the near UV and blue (300-500 nm) region of thespectrum.

None of the foregoing literature discloses fluorescent rigidized dyecompounds that are capable of producing strong fluorescence in the greento orange region of the spectrum and also contain functional groupsand/or solubilizing groups which render the dye suitable for covalentlabelling, in particular to biological molecules and other targetmaterials.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the absorption spectra for a solution of thedye of Example 1 as compared to protein labeled with the dye in Example1.6;

FIG. 2 compares the rigidized dye structure of the dye of Example 1 withthe structures of two open chain dyes.

FIG. 3 is a graph comparing the spectral properties of the three cyanine3 dyes of FIG. 2;

FIG. 4 is a graph showing the emission spectra of the three cyanine dyesof FIG. 2 when excited at 514 nm;

FIG. 5 shows the results of photo bleaching the three dyes of FIG. 2when exposed to laser;

FIG. 6 is a graph showing the result of a peptide polarization bindingassay according to Example 12.2;

FIG. 7 is a graph showing the results of the nucleic acid FREThybridization assay of Example 13.3;

FIG. 8 is a bar graph showing the results of a protein: DNA directintensity binding assay according to the procedures of Example 14.2;

FIG. 9 is a bar graph showing the results of a protein: DNA FRET bindingassay of Example 15.2; and

FIG. 10 is a graph plotting specific polarization readings againstconcentration of unlabeled ligand following Example 16.2.

The present invention provides bright, highly fluorescent dye compoundswhich absorb and emit in the 450-600 nm region of the spectrum. Theyhave rigid structures which are based on the trimethine cyaninechromophore and confer high quantum yields of fluorescence. Moreover,they can contain functional or reactive groups which may be used tocovalently react with suitable groups on target materials such asbiological molecules, and other materials. They are pH insensitive andthus they extend the range of useful fluorescent labelling reagentswhich can be used in fluorescent detection applications.

Accordingly, the present invention provides compounds of formula (2):

optionally substituted by groups R²-R⁹, wherein groups R⁶, R⁷, R⁸ and R⁹are attached to the rings containing X and Y or, optionally are attachedto atoms of the Z^(a) and Z^(b) ring structures;

R² to R⁹ which are the same or different include —R¹⁰ and -L-R¹⁰ whereR¹⁰ is selected from neutral groups that reduce water solubility, polargroups that increase water solubility, functional groups that can beused in labelling reactions, reactive groups, electron donating andwithdrawing groups that shift the absorption and emission wavelengths ofthe fluorescent molecule, lipid and hydrocarbon solubilising groups, andL is selected from the group consisting of a straight or branchedC₁₋₂₀alkyl chain, a C₂₋₂₀ monoether or polyether and a C₂₋₂₀ atom chaincontaining up to four secondary amide linkages;

R¹ is selected from hydrogen, aryl, heteroaryl, cyano, nitro, aldehyde,halogen, hydroxy, amino, quaternary amino, acetal, ketal, phosphoryl,sulphydryl, water-solubilizing groups, and alkyl groups optionallysubstituted by amino, C₁-C₄ alkyl-substituted amino, quaternary amino,carbonyl including aldehyde and ketone, acetal, ketal, halo, cyano,aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate, amide,nitro, and groups reactive with amino, hydroxyl, aldehyde, phosphoryl,or sulphydryl groups;

A is selected from O, S and NR¹¹ where R¹¹ is the substituted aminoradical:

where R′ is selected from hydrogen, a C₁₋₄ alkyl and aryl and R″ isselected from C₁₋₁₈ alkyl, aryl, heteroaryl, an acyl radical having from2-7 carbon atoms, and a thiocarbamoyl radical.

X and Y may be the same or different and are selected from bis-C₁-C₄alkyl and C₄-C₅ spiro alkyl substituted carbon, oxygen, sulphur,selenium, CH═CH, and N—W wherein N is nitrogen and W is selected fromhydrogen, a group —(CH₂)_(n)R¹² where n is an integer from 1 to 26 andR¹² is selected from hydrogen, amino, aldehyde, acetal, ketal, halo,cyano, aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate,substituted amino, quaternary amino, nitro, primary amide, substitutedamide, and groups reactive with amino, hydroxyl, carbonyl, phosphoryl,and sulphydryl groups;

Z^(a) and Z^(b) each represent a bond or the atoms necessary to completeone, two fused or three fused aromatic rings each ring having five orsix atoms, selected from carbon atoms and, optionally, no more than twooxygen, nitrogen and sulphur atoms;

provided that when X and Y are other than carbon, at least one of R¹-R⁹comprises a reactive group for covalent reaction with a functional groupon a target material or comprises a functional group for covalentreaction with a reactive group on a target material, or,when X and Y are different and are selected from O and Se, at least oneof R¹-R⁹ is other than hydrogen, methyl, phenyl or naphthyl.

Preferred R¹⁰ groups are selected from: hydrogen, halogen, amide, C₁-C₆alkoxy, nitro, cyano, aryl, heteroaryl, sulphonate, quaternary ammonium,guanidinium, hydroxyl, phosphate, phosphonate, optionally substitutedamino, azido, sulphydryl, carboxyl, carbonyl, reactive groups, forexample, succinimidyl ester, isothiocyanate, anhydride, haloacetamide,maleimide, sulphonyl halide, phosphoramidite, acid halide, alkylimidate,hydrazide and carbodiimide; and groups reactive with amino, hydroxyl,aldehyde, phosphoryl, or sulphydryl groups.

Preferably R¹ is selected from hydrogen, aryl, heteroaryl, cyano,halogen, alkyl groups of twenty-six carbon atoms or less and —(CH₂)_(n)Qwhere 1<n<26 and Q is selected from amino, aldehyde, sulphydryl,hydroxyl and groups reactive with amino, hydroxyl, aldehyde, phosphoryl,or sulphydryl groups and R², R³, R⁴ and R⁵ are hydrogen.

Suitably R¹² is selected from hydrogen, amino, sulphonate, carboxylate,aryl, hydroxyl, and groups reactive with amino, hydroxyl, carbonyl,phosphoryl, or sulphydryl groups.

Bis-substituted carbon includes bis C₁-C₄ alkyl groups and C₄-C₅ spiroalkyl groups.

Alkyl is a straight or branched chain alkyl group containing from 1-26carbon atoms, suitably containing from 1-12 carbon atoms, preferablyfrom 1-6 carbon atoms.

Aryl is an aromatic substituent containing one or two fused aromaticrings containing 6-10 carbon atoms, for example phenyl or naphthyl. Thearyl may be optionally and independently substituted by one or moregroups selected from groups —R¹⁰ and -L-R¹⁰ as hereinbefore defined.

Heteroaryl is a mono- or bicyclic 5-10 membered aromatic ring systemcontaining at least one and no more than 3 heteroatoms which may beselected from N, O and S. The heteroaryl may be optionally andindependently substituted by one or more groups selected from groups—R¹⁰ and -L-R¹⁰ as hereinbefore defined.

Aralkyl is a C₁-C₆ alkyl group substituted by an aryl or heteroarylgroup.

Halogen and halo-groups are those selected from fluorine, chlorine,bromine and iodine.

Specific examples of the groups R¹-R⁹ and R¹¹ and the groups with whichthose R-groups will react are provided in Table 1. In the alternative,the R¹-R⁹ and R¹¹ may be the functional groups of Table 1 which wouldreact with the reactive groups of a target molecule.

TABLE 1 Possible Reactive Substituents and Sites Reactive TherewithReactive Groups Corresponding Functional Groups succinimidyl estersprimary amino, secondary amino, hydroxyl anhydrides primary amino,secondary amino, hydroxyl acyl azides primary amino, secondary aminoisothiocyanates, isocyanates amino, thiol, hydroxyl sulphonyl chlorides,sulphonyl amino, hydroxyl fluorides substituted hydrazines, aldehydes,ketones substituted hydroxylamines acid halides amino, hydroxylhaloacetamides, maleimides thiol, imidazoles, hydroxyl, aminocarbodiimides carboxyl groups phosphoramidite hydroxyl

In addition to those groups listed in Table 1, a number of other groupsare possible as reactive substituent in the R¹-R⁹ and R¹¹ positions ofthe compounds of the present invention. For example, the reactive groupswhich are especially useful for labelling target components withavailable amino and hydroxy functional groups include:

where n=0 or an integer from 1-10 and at least one of R¹³ or R¹⁴ is aleaving group such as I, Br, or Cl.

Specific examples of possible R¹-R⁹ and R¹¹ groups that are especiallyuseful for labelling target components with available sulphydrylfunctional groups include:

where n=0 or an integer from 1-10 and R¹⁵ is a leaving group such as Ior Br.

Specific examples of possible R¹-R⁹ and R¹¹ functional groups that areespecially useful for labelling target components by light-activatedcross linking include:

For the purpose of increasing water solubility or reducing unwantednon-specific binding of the fluorescently-labelled component toinappropriate components in the sample or to reduce interactions betweentwo or more reactive chromophores on the labelled component which mightlead to quenching of fluorescence, the R¹-R⁹ and R¹¹ functional groupscan be selected from the well known polar and electrically chargedchemical groups. Examples of such groups are -E-F- where F is hydroxy,sulphonate, sulphate, carboxylate, substituted amino or quaternaryamino, and where E is a spacer group such as —(CH₂)_(n)— where n is 0-6.Useful examples of -E-F groups include C₁₋₆ alkyl sulphonates, such as—(CH₂)₃SO₃ ⁻ and —(CH₂)₄—SO₃.

Exemplary compounds of the present invention which demonstrate thecapability for adjusting fluorescence colour, water solubility, and theposition of the reactive or functional group are as follows:

i) 6,7,9,10-Tetrahydro-2,14-carboxymethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a;3′2′-a′]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound I);ii)8,9,11,12-Tetrahydro-3,17-disulphonato-20,20,22,22-tetramethyl-9aH,10aH-bisbenz[e]indolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-7-ium(Compound II);iii)6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound II);iv)6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium,glycinamide (Compound IV);v)6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium,N-(2-aminoethylcarboxamide) (Compound V);vi)6,7,9,10-Tetrahydro-2-(N-formyl)aminomethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VI);vii)6,7,9,10-Tetrahydro-2-hydroxyethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VII);viii)6,7,8,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-benzothiazolenine-indolenine-[3,2-a]-benzthiazolyl[3′2′-a]-pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VIII);ix)6,7,8,8a,9,10-Hexahydro-2,14-disulphonato-8-(4-carboxy-anilino)-16,16,18,18-tetramethyl-7aH-bis-indolinium[3,2-a;3′2′-a′]pyrido[3,2-c;5,6-c′]dipyridin-5-ium(Compound IX);x)6,7,9,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-quinolino-indolenium-[3,2-a,3′2′-a]-pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound X).

The groups provided herein are not meant to be all-inclusive of thosegroups which can be incorporated at the R sites of the compounds of thepresent invention. It will be understood that there are various othergroups which will react with groups on material that is to be labelledby the compounds of the present invention. Compounds produced by theincorporation of such other groups at the R¹-R⁹ and R¹¹ positions areintended to be encompassed by the present invention.

The compounds of the present invention may be used in numerousbiological and non-biological applications. With respect tonon-biological applications, compounds of the present invention havingone or more uncharged groups at the R¹-R⁹ and R¹¹ positions, forexample, C₁₋₂₆ alkyl and aryl moieties may be dissolved in non-polarmaterials to provide fluorescent properties to those materials. Suchnon-polar materials include, for example, paints, polymers, waxes, oils,inks and hydrocarbon solvents. Another non-biological application of thepresent invention is to dissolve compounds of the present inventionhaving one or more charged and/or polar groups at the R¹-R⁹ and R¹¹positions in polar solvents or other materials such as, for example,water, ethylene glycol, methyl alcohol, or a mixture of water and methylalcohol. Such charged R-groups include, for example, —NR₃ ⁺, —SO₃ ⁻,—PO₃ ⁻ and —COO⁻, while such polar R-groups include, for example,hydroxyl groups. With respect to biological applications, biologicalmolecules may be non-covalently labelled using the present complexes.

For example, complexes of the present invention wherein at least one ofR¹-R⁹ and R¹¹ contains a charge, for example, quaternary amino, may beused to non-covalently bind to charged biological molecules such as, forexample, DNA and RNA. In addition, compounds of the present inventionwherein at least one of R¹-R⁹ and R¹¹ is an uncharged group, forexample, a long chain alkyl, may be used to bind to uncharged biologicalmolecules such as, for example, biological lipids.

Alternatively, the compounds of the present invention may contain apolymerizable group suitable for the formation of a polymer containingthe complex. Suitable polymerizable groups are selected from acrylate,methacrylate, acrylamide, vinyl and styryl. Polymerization may becarried out with a suitably derivatized compound of this invention usedin conjunction with a second polymerizable monomer starting material,such as styrene or vinyltoluene, to form a copolymer containing thefluorescent compound. Alternatively the fluorescent compounds of theinvention need not have a polymerisable group, for example, the compoundmay be incorporated during polymerisation or particle formation or maybe absorbed into or onto polymer particles.

The dyes of the present invention can also be used as laser dyesaccording to the procedures set forth in U.S. Pat. No. 4,916,711 toBoyer and Morgan. Laser dyes must be fluorescent, must have a quantumyield greater than 0.56 or 0.57 and must be reasonably photostable. Thecompounds of the present invention satisfy each of these requirements.Further the dyes of the present invention can be used as textile dyes,photographic dyes and as organic conductors.

The compounds of the present invention may also be used to covalentlylabel a target material to impart fluorescent properties to the targetmaterial. Covalent labelling using the compounds of the presentinvention may be utilized either in a biological or a non-biologicalapplication. Examples of target materials that may be labelled innon-biological applications include, for example, cellulose-basedmaterials (including, for example, papers), textiles, petroleum-basedproducts, photographic films, glasses, polymers and gel filtration andchromatography media.

Covalent labelling using compounds of the present invention may beaccomplished with a target having at least one functional or reactivegroup as defined hereinbefore. The target may be incubated with anamount of a compound of the present invention having at least one ofR¹-R⁹ and R¹¹ that includes a reactive or functional group ashereinbefore defined that can covalently bind with the functional orreactive group of the target material. The target material and thecompound of the present invention are incubated under conditions and fora period of time sufficient to permit the target material to covalentlybond to the compound of the present invention.

R¹-R⁹ and R¹¹ can be chosen so that the compounds of the presentinvention react with different target compounds and/or to have differentspectral properties, thereby providing a number of related compoundswhich can be used in multiplex analyses wherein the presence andquantity of various compounds in a single sample must be differentiatedbased on the wavelengths and intensities of a number of detectedfluorescence emissions. The compounds of the present invention may bemade soluble in aqueous, other polar, or non-polar media containing thematerial to be labelled by appropriate selection of R-groups.

The invention also relates to labelling methods wherein the compounds ofthe present invention including at least one reactive group at the R¹-R⁹and R¹¹ positions covalently react with amino, hydroxyl, aldehyde,phosphoryl, carboxyl, sulphydryl or other reactive groups on targetmaterials. Such target materials are include, but are not limited to thegroup consisting of antibody, lipid, protein, peptide, carbohydrate,nucleotides which contain or are derivatized to contain one or more ofan amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate andthiophosphate groups, and oxy or deoxy polynucleic acids which containor are derivatized to contain one or more of an amino, sulphydryl,carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups,microbial materials, drugs, toxins, particles, plastics or glasssurfaces and polymers. Compounds of the present invention may also beused for coupling to additional fluorescent or non-fluorescent compoundsfor use in fluorescence resonance energy transfer complexes of the typedescribed in EPA 747700 or for fluorescence polarisation or fluorescencequenching-based applications.

In addition to the foregoing single-step labelling process, the presentinvention also relates to two-step labelling processes in which, in afirst step, a compound of the present invention covalently reacts withand thereby labels a primary component, such as an antibody. In a secondor staining step of the two-step procedure, the fluorescently labelledprimary component is then used as a probe for a secondary component,such as an antigen for which the antibody is specific. When the targetof the so-labelled antibodies is a cell, the second step of theprocedure may be used to determine the amount of labelled antibodieswhich are attached to that type of cell by determining the intensity ofthe fluorescence of the cells. By this two-step procedure, monoclonalantibodies and other components covalently labelled in the first stepwith the fluorescent compounds of the present invention could be used asantigen probes.

The compounds of the present invention can be used to determine theconcentration of a particular protein or other component in a system. Ifthe number of reactive groups on a protein which can react with a probeis known, the fluorescence per molecule can be known and theconcentration of these molecules in the system can be determined by thetotal fluorescence intensity of the system. This particular method canbe used to measure the concentration of various labelled analytes usingmicrotitre plate readers or other known immunofluorescence detectionsystems.

The compounds of the present invention are also useful in assaymethodologies that employ fluorescent labels for the detection andmeasurement of analytes, using for example, fluorescence resonanceenergy transfer (FRET) based methods, fluorescence lifetime, or by meansof fluorescence polarization measurements.

The use of fluorescence resonance energy transfer dye pairs inbiological systems is well known and they have been used in thedetection of binding events or cleavage reactions in assays which employFRET. Examples of such assays include equilibrium binding assays, (eg.immunoassays, nucleic acid hybridisation assays, protein binding assaysand hormone receptor assays) and enzyme assays, such as proteolyticcleavage assays, the cleavage of a DNA or RNA molecule by a nuclease, ora lipid by a lipase.

Binding assays utilising compounds of the present invention may beperformed by binding one component of a specific binding pair with asecond component of the specific binding pair, the first component beinglabelled with a fluorescent donor dye according to the presentinvention, and the second component being labelled with a fluorescent(or quenching) acceptor dye, so as to bring about an energy transferrelationship between the first and second components, and detecting thebinding of the first and second components by measurement of the emittedfluorescence. Examples of specific binding pairs include, but are notrestricted to, antibodies/antigens, lectins/glycoproteins,biotin/(strept)avidin, hormone/receptor, enzyme/substrate or co-factor,DNA/DNA, DNA/RNA and DNA/binding protein. It is to be understood that inthe present invention, any molecules which possess a specific bindingaffinity for each other may be employed, so that the dyes of the presentinvention may be used for labelling one component of a specific bindingpair, which in turn may be used in the detection of binding to the othercomponent.

The dyes of the present invention may also be used in an enzyme cleavageassay format, in which the enzyme substrate, for example a peptide,comprises two components, one of which is labelled with a fluorescentdonor dye of the present invention, the second being labelled with afluorescent (or quenching) acceptor dye and being attached to thesubstrate in an energy transfer relationship on either side of thesubstrate bond to be cleaved. A known or a putative enzyme inhibitorcompound may be optionally included in the reaction mixture. Cleavage ofthe substrate by the enzyme results in separation of the donor andacceptor dyes, resulting in a loss of resonance energy transfer and achange in the fluorescence emission of the donor and acceptor species.

Suitable fluorescent acceptor dyes that can be combined with the dyes ofthe present invention to form energy transfer dye pairs include therhodamine and cyanine dyes. Particularly preferred are the cyanine dyes,including Cy5(1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-5,5′-disulphonato-dicarbocyanine),Cy5.5(1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-4,5,4′,5′-(1,3-disulphonato)-dibenzo-dicarbocyanine)and Cy7(1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-5,5′-disulphonato-tricarbocyanine).A suitable quenching acceptor dye is DABCYL(4-(4-dimethylaminophenyl)azobenzoic acid).

The dyes of the present invention may also be used in binding assays orin enzyme cleavage assays, utilising fluorescence polarizationmeasurements. In a binding assay format, the assay of an analyte in asample may be performed by providing a specific binding partner for theanalyte, the specific binding partner being labelled with a dyeaccording to the present invention, measuring the fluorescencepolarization of the labelled specific binding partner, contacting theanalyte with the labelled specific binding partner under conditionssuitable for binding the analyte to form an analyte-specific bindingpartner complex and measuring the fluorescence polarization of thelabelled analyte-specific binding partner complex to determine theextent of binding.

In the second format, an assay for the detection of enzyme activity maybe configured as follows. A reaction mixture is prepared by combining aprotease enzyme and a fluorogenic substrate labelled with a dyeaccording to the present invention. A known or a putative inhibitorcompound may be optionally included in the reaction mixture. Cleavage ofthe substrate by the enzyme results in the production of labelledfragments. The progress of the reaction is monitored by observing thechange in fluorescence polarization.

The fluorescent compounds of the present invention can also be used in adetection method wherein a plurality of the fluorescent compounds arecovalently attached to a plurality of different primary components, suchas antibodies, each primary component being specific for a differentsecondary component, such as an antigen, in order to identify each of aplurality of secondary components in a mixture of secondary components.According to this method of use, each of the primary components isseparately labelled with a fluorescent compound having a different lightabsorption and emission wavelength characteristic compared with the dyemolecules used for labelling the other primary components. The so-calledprimary components are then added to the preparation containingsecondary components, such as antigens, and the primary components areallowed to attach to the respective secondary components for which theyare selective.

Any unreacted probe materials may be removed from the preparation by,for example, washing, to prevent interference with the analysis. Thepreparation is then subjected to a range of excitation wavelengthsincluding the absorption wavelengths of particular fluorescentcompounds. A fluorescence microscope or other fluorescence detectionsystem, such as a flow cytometer or fluorescence spectrophotometer,having filters or monochrometers to select the rays of the excitationwavelength and to select the wavelengths of fluorescence is nextemployed to determined the intensity of the emission wavelengthscorresponding to the fluorescent compounds utilized, the intensity offluorescence indicating the quantity of the secondary component whichhas been bound with a particular labelled primary component. Knowntechniques for conducting multi-parameter fluorescence studies include,for example, multi-parameter flow cytometry.

In certain cases a single wavelength of excitation can be used to excitefluorescence from two or more materials in a mixture where eachfluoresces at a different wavelength and the quantity of each labelledspecies can be measured by detecting its individual fluorescenceintensity at its respective emission wavelength. If desired, a lightabsorption method can also be employed.

The detection method of the present invention can be applied to anysystem in which the creation of a fluorescent primary component ispossible. For example, an appropriately reactive fluorescent compoundcan be conjugated to a DNA or RNA fragment and the resultant conjugatethen caused to bind to a complementary target strand of DNA or RNA.Appropriate fluorescence detection equipment can then be employed todetect the presence of bound fluorescent conjugates.

The present invention also relates to the covalent reaction betweencompounds of the present invention, and amine, hydroxy, aldehyde,sulphydryl, phosphoryl or other known functional groups on materialssuch as, for example, proteins, peptides, carbohydrates, nucleic acids,derivatized nucleic acids, lipids, certain other biological molecules,biological cells, soluble polymers, polymeric particles, polymersurfaces, polymer membranes, glass surfaces and other particles andsurfaces. Because detecting fluorescence involves highly sensitiveoptical techniques, the presence of these dye “labels” can be detectedand quantitated even when the label is present in very low amounts.Thus, the dye labelling reagents can be used to measure the quantity ofa material that has been labelled.

Compared with, for example, the fluoresceins, the rigidized trimethinecyanines of the present invention are particularly photostable and areinsensitive to pH changes between pH2 and pH10. The compounds of thepresent invention maximally absorb and emit light at wavelengths between450 and 600 nm (green to orange region of the spectrum) and aretherefore alternatives to Texas-Red, rhodamine, tetramethylrhodamine,X-rhodamine, BODIPY and fluorescein.

The present invention also provides a process for the preparation of acompound of formula (2) which comprises treating a compound of formula(A):

optionally substituted by groups R²-R⁹, wherein X, Y, Z^(a), Z^(b) andgroups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are as defined above and Ris methyl or ethyl, in mild acid solution, such as in acetic acid.Suitably the reaction mixture is heated under refluxing conditions,whereupon the rigidized carbocyanine dye precipitates from solution.Alternatively the reaction may be carried out in a stronger mineral acidsolution, such a sulphuric acid at lower temperatures, for exampleambient temperature. It may be advantageous to include in the reactionmixture, a solvent such as chloroform.

In the case of amino and hydrazino substituted carbocyanine dyes of thepresent invention, these may be prepared from intermediates of generalstructure (A) by including the appropriate amine or hydrazino derivativein the acid solution used for preparing the rigidized dye.

Symmetrical compounds of structure (A) wherein X and Y are the same andstructures Z^(a) and Z^(b) are the same may be prepared by reacting acompound of structure (B):

optionally substituted with groups R², R³, R⁶ and R⁷ wherein groups,Z^(a) and X, R², R³, R⁶ and R⁷ are as hereinbefore defined and R ismethyl or ethyl, with an appropriate ortho ester such a ethylorthoformate in a suitable solvent medium to prepare the non-rigidizedtrimethine. The reaction is suitably carried out in solution in asolvent such as pyridine by heating under reflux. By suitablysubstituting the ortho ester, the central or meso carbon atom of theconjugated trimethine chain may be substituted with a variety ofsubstituents such as are represented by the group R¹. For example,replacement of the ethyl orthoformate in the reaction mixture with ethylorthoacetate will produce a trimethine cyanine dye in which the mesohydrogen is replaced with a methyl group.

Asymmetric compounds of structure (A) wherein X and Y are different maybe prepared by reacting a compound of formula (B), optionallysubstituted with groups R⁵ and R⁶ wherein groups R², R³, R⁵, R⁶, Z^(a)and X are as hereinbefore defined with a compound of structure (C):

optionally substituted by groups R⁸ and R⁹ wherein groups R¹, R⁴, R⁵,R⁸, R⁹, Y and Z^(b) are as hereinbefore defined, R is an alkyl groupsuch as methyl or ethyl, R^(a) is an acyl radical, such as acetyl,propionyl and benzoyl and Rb is hydrogen, an alkyl radical such asmethyl or ethyl, or an aryl radical such as phenyl. The reaction issuitably carried out in a 1:1 molar proportion in acetic anhydridesolution.

Intermediate compound (B) may be prepared by reacting the hydrohalideacid salt of the appropriate heterocyclic base of formula (D):

optionally substituted by groups R⁶ and R⁷, wherein X, Z^(a), R⁶ and R⁷are as defined above with a compound of formula (E):

wherein R² and R³ are as hereinbefore defined. The reaction isadvantageously carried out with reagent (E) in excess and in an inertsolvent of moderate polarity that dissolves both reagents, but which isnot a solvent for the reaction product. Examples of such media aresolvents such as acetonitrile. The reaction is suitably carried out atan elevated temperature, suitably 70° C. An acid such as acetic acid maybe added to the reaction mixture to facilitate the reaction. As aspecific example the hydrobromide salt of(2,3,3-trimethyl-3H-indol-5-yl)-acetic acid prepared by the method ofSouthwick et al (Org. Prep. Proceed. Int. 20, 279-84, 1989) is reactedwith acrolein diethyl acetal in acetonitrile containing acetic acid assolvent. The reaction is suitably carried out at a temperature of 70° C.

Intermediates of formula (C) may be prepared by reaction of a compoundof structure (B) containing a methyl substituent in the 2-position witha formamidine of formula (F):

wherein R¹ and R^(b) are as hereinbefore defined and R^(c) is phenyl orsubstituted phenyl. Suitably the reaction is carried out by condensingthe quaternary salt of structure (B) with a 1.5 molar excess of theformamidine using an acid condensing agent, for example aceticanhydride, propionic anhydride, or glacial acetic acid. Acetic anhydrideis a particularly preferred condensing agent for the reaction.Alternatively, the condensation reaction may be performed without anyaddition; however acid condensation is to be preferred for theproduction of rigidized trimethine cyanine dyes substituted at thecentral or meso carbon atom of the trimethine chain. See, for example,British Patent No. 412309.

Precursor compounds of formula such as (D) may be prepared by methodswell known to those skilled in the area. See for example U.S. Pat. No.4,981,977, the entire disclosure of which is incorporated by reference.

It will be readily appreciated that certain compounds of formula (2) maybe useful as intermediates for conversion to other compounds of theformula (2) by methods well known to those skilled in the art. Likewise,certain of the intermediates may be useful for the synthesis ofderivatives of formula (2). The compounds of the present invention maybe synthesized by the methods disclosed herein. Derivatives of thecompounds having a particular utility are prepared either by selectingappropriate precursors or by modifying the resultant compounds by knownmethods to include functional groups at a variety of positions. Asexamples, the complexes of the present invention may be modified toinclude certain reactive groups for preparing a fluorescent labellingreagent, or charged or polar groups may be added to enhance thesolubility of the compound in polar or nonpolar solvents or materials.As examples of conversions an ester may be converted to a carboxylicacid or may be converted to an amido derivative.

The following are specific examples of the synthesis of compounds of thepresent invention and observed spectral data for those compounds.

EXAMPLE 16,7,9,10-Tetrahydro-2,4-carboxymethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a;3′2′-a′]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(R-Cy3.12.OH; Compound I)

1.1 5-Carboxymethyl-2,3,3-trimethylindoline

5-Carboxymethyl-2,3,3-trimethylindoline was prepared either by themethod of Southwick et al, Org. Prep. Proceed. Int., 20, 274-84, (1989),or alternatively as described below.

To a stirred solution of 4-aminophenylacetic acid (5 g, 33.1 mmol) in a3:2 water: conc. HCl (33 ml) solvent mixture at <0° C. was addeddropwise a cooled (<0° C.) solution of sodium nitrite (2.7 g, 39 mmol)in water (43 ml). The reaction mixture was then maintained at thereduced temperature for a further 30 minutes. A saturated aqueoussolution of sulphur dioxide (140 ml) was added and the reaction mixturewarmed to ambient temperature over 1 hour, then warmed for a furtherhour at 70° C. The reaction mixture was cooled rapidly and the solventremoved in vacuo. The yellow hydrazine intermediate product obtained wasredissolved in acetic acid (54 ml) and potassium acetate (7.05 g, 71.8mmol) and methyl-isopropyl ketone (6.84 g, 79.4 mmol) added at ambienttemperature. After 30 minutes the reaction mixture was warmed to 90° C.and stirred for a further 2 hours. The reaction mixture was cooled andthe reaction solvent removed in vacuo. The product was dissolved indichloromethane (100 ml) and washed with water (2×50 ml). The organicphase was dried over MgSO₄, filtered and concentrated in vacuo.5-ethoxycarbonyl-2,3,3-trimethylindolenine was obtained as a red solid(4.8 g, 67%). No purification was required; m/z (Maldi): 217.

1.2 1-(3,3-Diethoxypropyl-5-carboxymethyl-2,3,3-trimethylindolenine,ethyl ester

To a stirred solution of 5-carboxymethyl-2,3,3-trimethylindolenine (2 g,9.3 mmol) in ethanol (40 ml) at ambient temperature was addedhydrobromic acid (3.16 ml of 48% aqueous solution). After 1 hour thereaction solvent was removed in vacuo. The hydrobromide salt wasredissolved in acetonitrile (40 ml) and acetic acid (400 ml) andacrolein diethyl acetal (18.17 g, 140 mmol) added. The reaction mixturewas warmed to 70° C. for 20 minutes. The solution was cooled and thereaction solvent removed in vacuo. The product was purified by HPLC on aRainin Dynamax C18, 8 μm column using a 10-100% gradient elution ofwater/acetonitrile (containing 0.1% TFA) over 60 minutes at 20 ml/min.The product was obtained as a green oil (1.24 g, 36%); m/z (FAB⁺):376.2.

1.35,5′-Dicarboxymethyl-1,1′-di-(3,3-diethoxypropyl)-indocarbocyanine-ethylester

To a stirred solution of1-(3,3-diethoxypropyl-5-carboxymethyl-2,3,3-trimethylindolenine, ethylester (356 mg, 0.95 mmol) in pyridine (10 ml) at 120° C. was addeddropwise, triethyl orthoformate (98 mg, 66 mmol) over 30 minutes. After2 hours the reaction mixture was cooled. The product was purified byHPLC on a Rainin Dynamax C18, 8 μm column using a 10-100% gradientelution of water/acetonitrile (containing 0.1% TFA) over 60 minutes at20 ml/min. The product was obtained as a pink solid (251 mg, 35%); λmax:555 nm; m/z (FAB⁺): 761.4.

1.46,7,9,10-Tetrahydro-2,14-carboxymethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium

To a stirred solution of5,5′-carboxymethyl-1,1-di-(3,3-diethoxypropyl)-indocarbocyanine, ethylester (100 mg, 0.132 mmol) in chloroform (10 ml) at ambient temperaturewas added 50% aqueous sulphuric acid (2 ml). After 30 minutes thereaction solvent was diluted with chloroform (10 ml) and washed withwater (3×10 ml). The organic phase was dried over NaSO₄, filtered andconcentrated in vacuo. The product was purified by HPLC on a RaininDynamax C18, 8 μm column using a 10-100% gradient elution ofwater/acetonitrile (containing 0.1% TFA) over 60 minutes at 20 ml/min.The product was obtained as a pink solid (65 mg, 90%); λmax 565 nm; m/z;m/z (FAB⁺): 539.2.

1.56,7,9,10-Tetrahydro-2,14-carboxymethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium,N-hydroxysuccinimidyl ester

To a mixture of O—(N-succinimidyl-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate (5 mg, 0.012 mmol), and N,N′-diisopropylethylamine(4.08 mg, 0.032 mmol) in dimethylsulphoxide (500 μl) at ambienttemperature was added6,7,9,10-tetra-2,14-carboxymethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(5 mg, 0.0089 mmol). The reaction mixture was stirred for 1 hr.Conversion to the N-hydroxysuccinimidyl ester derivative was confirmedby mass spectroscopy and HPLC using a Phenomenex Jupiter C18 10 μmcolumn.

1.6 Protein Labelling Procedure

A stock solution of the N-hydroxysuccinimidyl ester of Compound I wasprepared in dry DMF (1 mg active ester/100 μl). Sheep IgG (1 mg, 6.45mmol) was dissolved in 250 μl buffer solution (pH 9.4) and the desiredamount of dye was added during vigorous vortex mixing. Unconjugated dyewas separated from the labelled protein by gel permeation chromatography(0.7×20 cm column of Sephadex G-50) using pH 7 buffer solution aseluant. Absorption spectra of the labelled antibody solution wasrecorded (see FIG. 1). Dye to protein ratio for the sample wasdetermined using an equation below with measured values of absorbance ofthe labelled dye at 560 nm and the absorbance of protein at 280 nm.

$\frac{D}{P} = \frac{A_{dye} \times E_{prot}}{\left( {A_{280} - {XA}_{dye}} \right) \times E_{dye}}$

The factor X in the denominator accounts for the dye absorption at 280nm which is a % of the absorption of the dye at its maximum absorption(A_(dye)). The value of X is 0.17 for a rigid dye.

1.7 Comparison of Spectral Properties: Rigidized Cy3 (Compound I) andOpen Chain Cy3

The spectral properties of the rigidized dye were compared with theknown open chain Cy3.18.OH and Cy3.10.OH dyes (FIG. 2). The absorptionabsorption and emission spectra are shown in FIGS. 3 and 4. Theabsorption maxima of the rigid dye shifted to the red by 12 nm inmethanol and as expected it is 10-12 time brighter that thenon-rigidized indocyanines. The results are shown in Table 2 below.

TABLE 2 Dye in Methanol λ_(max) ε_(max) QY (Φ) R-Cy3•12•OH (Compound I)565 584 0.8 Cy3•18•OH 555 570 0.09 Cy3•10•OH 555 578 0.08

Photo-bleaching of the dyes was studied under identical conditions.Samples of equal concentrations of dyes (3 ml of 1.5×10⁻⁵ mmol solutionin water) were exposed to a laser line 514 nm (30 mV, 1 cm diam. beam)and absorption was recorded in a few minutes of interval for 50 minutes.The results are shown in FIG. 5. Rigidized dye is expected to bleachfaster. However, because of its high quantum yield, it is expected thatthe rigidized dye will be suitable for flow cytometer and other imagingexperiments where the sample is exposed for short time periods.

EXAMPLE 28,9,11,12-Tetrahydro-3,17-disulphonato-20,20,22,22-tetramethyl-9aH,10aH-bisbenz[e]indolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-7-ium(Compound II)

2.1 6-Sulphonato-2,3,3-trimethyl-1H-benz[e]indolenine

A stirred solution of 2,3,3-trimethyl-1H-benz[e]-indolenine (100 g, 478mmol) in concentrated sulphuric acid (500 ml) was heated at 180° C.After 2 hours the solution was cooled to ambient temperature, thenpoured onto ice. The reaction mixture was made basic by adding 50%sodium hydroxide (3000 ml). The resulting precipitate was filtered,recrystallised from water and dried. The product was obtained as a whitesolid (7.25 g, 54%).

2.21-(3,3-Diethoxypropyl)-6-sulphonato-2,3,3-trimethyl-1H-benz[e]indolenine

1-(3,3-Diethoxypropyl)-6-sulphonato-2,3,3-trimethyl-1H-benz[e]indoleninewas prepared by reaction of6-sulphonato-2,3,3-trimethyl-1H-benz[e]indolenine (25 mg, 0.0087 mmol)with acrolein diethyl acetal (169 mg, 1.3 mmol) and acetic acid (10 P)in acetonitrile (2 ml) by an analogous method to that described inSection 1.2. The compound was not purified, as decomposition wasobserved. The product was obtained as a pale yellow oil.

2.36,6′-Disulphonato-1,1′-di-(3,3-diethoxypropyl)-benz[e]indo-carbocyanine

6,6′-Disulphonato-1,1-di-(3,3-diethoxypropyl)-benz[e]indocarbocyaninewas prepared by reaction of1-(3,3-diethoxypropyl)-6-sulphonato-2,3,3-trimethyl-1H-benz[e]indoleninewith triethyl orthoformate (51.2 mg, 0.035 mmol) in pyridine (5 ml) byan analogous method to that described in Section 1.3. The compound waspurified by HPLC on a Phenomenex Jupiter C18, 10 m column using 0-100%gradient elution of water/acetonitrile (containing 0.1% TFA) over 30minutes at 4 ml/min. The product was obtained as a pink/purple solid;λ_(max)(MeOH) 580 nm, m/z (Maldi): 852.

2.48,9,11,12-Tetrahydro-3,17-disulphonato-20,20,22,22-tetramethyl-9aH,10aH-bisbenz[e]indolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-7-ium

8,9,11,12-Tetrahydro-3,17-disulphonato-20,20,22,22-tetramethyl-9aH,10aH-bisbenz[e]indolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-7-iumwas prepared by reaction of6,6′-disulphonato-1,1-di-(3,3-diethoxypropyl)-benz[e]indocarbocyanine (3mg, 0.0035 mmol) in chloroform (5 ml) and 50% sulphuric acid (1 ml) byan analogous method to that described in section 1.4. The compound waspurified by HPLC on a Phenomenex Jupiter C18, 10 μm column using 0-100%gradient elution of water/acetonitrile (containing 0.1% TFA) over 30minutes at 4 ml/min. The product was obtained as a luminescentpink/purple solid; λmax (MeOH) 598 nm; m/z (Maldi): 684.

EXAMPLE 36,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound III)

3.1 5-Sulphonato-2,3,3-trimethylindolenine

To a stirred solution of 4-hydrazinobenzene sulphonic acid (68 g, 361mmol) in acetic acid (205 ml) at ambient temperature was added3-methyl-2-butanone (88.44 g, 1027 mmol). The reaction was heated underreflux. After 3 hours the solution was cooled and the resulting pinkprecipitate was filtered, washed with acetic acid (50 ml) and dried. Theproduct was redissolved in methanol (800 ml) and a solution of potassiumhydroxide (20.4 g, 364 mmol) in isopropanol (200 ml) was added. Theyellow solid obtained was filtered and dried (48 g, 56%); m/z (FAB⁺):240.

3.2 1-(3,3-Diethoxypropyl)-5-sulphonato-2,3,3-trimethylindolenine

1-Diethoxy propyl-5-sulphonato-2,3,3-trimethylindolenine was prepared byreaction of 5-sulphonato-2,3,3-trimethylindolenine potassium salt (1 g,3.88 mmol) with acrolein diethyl acetal (8.54 g, 65.6 mmol) and aceticacid (1 ml) in acetonitrile (40 ml) using an analogous method to thatdescribed in Section 1.2. The compound was purified by HPLC on a RaininDynamax C18, 8 μm column using 0-100% gradient elution ofwater/acetonitrile (containing 0.1% TFA) over 60 minutes at 20 ml/min.The product was obtained as a green oil (740 mg, 52%); m/z: (FAB⁺)370.1.

3.35-Carboxymethyl-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenine

To a stirred solution of5-carboxymethyl-1-(3,3-diethoxypropyl)-2,3,3-trimethylindolenine-ethylester (1.16 g, 3.09 mmol) in acetic anhydride (25 ml) was addedN,N′-diphenylformamidine (908 mg, 4.63 mmol). The reaction mixture waswarmed to 110° C. After 30 minutes the solution was cooled and thereaction solvent removed in vacuo. The product was purified by HPLC on aRainin Dynamax C18, 8 μm column using 10-100% gradient elution ofwater/acetonitrile (containing 0.1% TFA) over 60 minutes at 20 ml/min.The product was obtained as a pale brown oil (634 mg, 40%); m/z (FAB⁺):521.2.

3.45-Carboxymethyl-5′sulphonato-1,1′-di-(3,3-diethoxypropyl)-indocarbocyanine,ethyl ester

To a stirred solution of5-carboxymethyl-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenine(96 mg, 0.19 mmol) in 4.5:4.5:1 pyridine:acetic acid:acetic anhydride (5ml) at ambient temperature was added a solution of5-sulphonato-1-(3,3-diethoxypropyl)-2,3,3-trimethylindolenine (67.7 mg,0.19 mmol) in 4.5:4.5:1 pyridine:acetic acid:acetic anhydride (5 ml).The reaction mixture was warmed to 70° C. for 5 hours. The solution wascooled and the reaction solvent removed in vacuo. The product waspurified by HPLC on a Rainin Dynamax C18, 8 μm column using a 10-100%gradient elution of water/acetonitrile (containing 0.1% TFA) over 60minutes at 20 ml/min. The product was obtained as a pink solid (33 mg,24%); λmax (MeOH) 555 nm; m/z (FAB⁺): 755.3.

3.56,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium

6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-iumwas prepared by reaction of5-carboxymethyl-5′sulphonato-1-di-(3,3-diethoxypropyl)-indocarbocyanine,ethyl ester (33 mg, 0.044 mmol) in chloroform (10 ml) and 50% sulphuricacid (2 ml) by an analogous method to that described in Section 1.4. Theproduct was purified by HPLC on a Rainin Dynamax C18 column using a0-100% gradient elution of water/acetonitrile (containing 0.1% TFA) over60 minutes at 20 ml/min. The product was obtained as a pink solid (18.7mg, 76%); λmax (MeOH) 563 nm; m/z (FAB⁺): 561.

3.66,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium,N-hydroxysuccinimidyl ester

To a mixture of O—(N-succinimidyl-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate (5 mg, 0.012 mmol), and N,N′-diisopropylethylamine(4.08 mg, 0.032 mmol) in dimethyl sulphoxide (500 μl) at ambienttemperature was added6,7,9,10-tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(5 mg, 0.0089 mmol). The reaction was stirred for 1 hr. Conversion tothe N-hydroxysuccinimidyl ester derivative was confirmed by massspectroscopy and HPLC using a Phenomenex Jupiter C18 10 μm column.

EXAMPLE 46,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium,glycinamide (Compound IV)

To a mixture of O—(N-succinimidyl-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate (1 mg, 0.0024 mmol) and N,N′-diisopropylethylamine(0.82 mg, 0.0032 mmol) in dimethylformamide (100 ml) at ambienttemperature was added6,7,9,10-tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(1 mg, 0.0018 mmol). After 1 hour, glycine (0.2 mg, 0.0027 mmol) wasadded and the solution stirred for a further 3 hours. The product waspurified by HPLC on a Phenomenex Jupiter C 8, 10 μm column, using 0-100%gradient elution of water/acetonitrile (containing 0.1% TFA) at 4ml/min. The product was obtained as a pink solid (0.22 mg, 30%); m/z(Maldi): 618.

EXAMPLE 56,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium,N-(2-aminoethylcarboxamide) (Compound V)

To a mixture of O—(N-succinimidyl-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate (5 mg, 0.012 mmol) and N,N′-diisopropylethylamine(4.08 mg, 0.032 mmol) in dimethylformamide (500 ml) at ambienttemperature was added6,7,9,10-tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(5 mg, 0.0089 mmol). After 1 hour tert-butyl-N-(2-aminoethyl)-carbamate(1.4 mg, 0.0089 mmol) was added and the solution stirred for a further 2hours. The solvent was removed in vacuo. The product was dissolved in a95% aqueous trifluoroacetic acid solution and stirred for 2 hours. Theproduct was purified by HPLC on a Phenomenex Jupiter C18, 10 μm column,using gradient elution of acetonitrile/water (containing 0.1% TFA). Theproduct was obtained as a pink solid (1.6 mg, 30%); m/z (FAB⁺): 603.1.

EXAMPLE 66,7,9,10-Tetrahydro-2-(N-formyl)aminomethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VI)

6.1 5-Phthalimidomethyl-2,3,3-trimethylindolenine

To a stirred solution of 2,3,3,-trimethylindolenine (20 g, 126 mmol) inconcentrated sulphuric acid (100 ml) at ambient temperature was addedportionwise N-hydroxymethylphthalimide (20 g, 114 mmol). After 70 hoursthe reaction mixture was poured onto ice and made basic withconcentrated ammonium hydroxide. The resulting precipitate was filteredand dried. The product was obtained as a yellow solid (34.84 g, 87%).

6.2 5-Aminomethyl-2,3,3-trimethylindolenine

To a stirred solution of 5-phthalimidomethyl-2,3,3-trimethylindolenine(10 g, 31.4 mmol) in methanol (50 ml) at ambient temperature was addedhydrazine hydrate (13.1 g 409 mmol). After 20 hours a precipitate wasformed. The reaction mixture was adjusted to pH 1 with 6N HCl and thesolvent removed in vacuo. The solid obtained was suspended in 1N HCl andfiltered through celite. The filtrate was washed with dichloromethane(3×40 ml) and the aqueous phase adjusted to pH 12 with 6N NaOH, thenextracted with dichloromethane (3×40 ml). The organic phase was driedover Na₂SO₄, filtered and concentrated in vacuo to give a pale yellowsolid (4.98 g, 84%); m/z (Maldi): 188.

6.3 5-(N-Formyl)aminomethyl-2,3,3-trimethylindolenine

A stirred solution of 5-aminomethyl-2,3,3-trimethylindolenine (4.98 g,26.5 mmol) in methyl formate (30 ml) was refluxed under a nitrogenatmosphere for 22 hours. The solution was cooled and the solvent removedin vacuo. The product was obtained as pale brown oil (5.4 g, 94%); m/z(FAB⁺): 217.1.

6.41-(3,3-Diethoxypropyl)-5-(N-formyl)aminomethyl-2,3,3-trimethylindolenine

1-(3,3′-Diethoxypropyl)-5-(N-formyl)aminomethyl-2,3,3-trimethylindoleninewas prepared by reaction of5-(N-formyl)aminomethyl-2,3,3-trimethylindolenine (1 mg, 24.8 mmol) withacrolein diethyl acetal (9 g, 69.1 mmol) and acetic acid (1 ml) inacetonitrile (40 ml) by an analogous method to that described in Section1.2. The product was purified by HPLC on a Rainin Dynamax C18, 8 μmcolumn using a 0-100% gradient elution of water/acetonitrile (containing0.1% TFA) over 60 minutes at 20 ml/min The product was obtained as ayellow oil (1.10 mg, 69%); m/z (Maldi): 347.

6.51-(3,3-Diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-5-sulphonato-2,3,3-trimethylindolenine

1-(3,3-Diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-5-sulphonato-2,3,3-trimethylindoleninewas prepared by reaction of5-sulphonato-1-(3,3′diethoxypropyl)-2,3,3-trimethylindolenine (100 mg,0.27 mmol) [prepared as described in Section 3.2] withN,N′-diphenylformamidine (79 mg, 0.405 mmol) in acetic anhydride (20 ml)by an analogous method to that described in Section 3.3. The product wasnot purified, as decomposition was observed. The product was obtained asa yellow oil.

6.611-Di-(3,3′-diethoxypropyl)-5-(N-formyl)aminomethyl-5′-sulphonato-indocarbocyanine

To a stirred solution of5-sulphonato-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenine(37 mg, 0.072 mmol) in 4.5:4.5:1 pyridine:acetic acid:acetic anhydride(5 ml) at ambient temperature was added a solution of1-(3,3-diethoxypropyl)-5(N-formyl)aminomethyl-2,3,3-trimethylindolenine(25 mg, 0.072 mmol) in 4.5:4.5:1 pyridine:acetic acid:acetic anhydride(5 ml). The reaction mixture was warmed to 70° C. for 5 hours. Thesolution was cooled and the solvent removed in vacuo. The product waspurified by HPLC on a Rainin dynamax C18, 8 μm column using a 10-100%gradient elution of water/acetonitrile (containing 0.1% TFA) over 60minutes at 20 ml/min. The product was obtained as a pink solid (16 mg,15%); λmax (MeOH); 555 nm, m/z (FAB⁺): 726.1.

6.76,7,9,10-Tetrahydro-2-(N-formyl)aminomethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium

6,7,9,10-Tetrahydro-2-(N-formyl)aminomethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-iumwas prepared by reaction of1,1-Di-(3,3-diethoxypropyl)-5-(N-formyl)aminomethyl-5′-sulphonato-indocarbocyanine(5 mg, 0.0069 mmol) in chloroform (5 ml) and 50% sulphuric acid (1 ml)by an analogous method to that described in Section 1.4. The product waspurified by HPLC on a Rainin Dynamax C18, 8 μm column using a 0-100%gradient elution of water/acetonitrile (containing 0.1% TFA) over 60minutes at 20 ml/min. The product was obtained as a pink solid (3.3 mg,90%); λmax (MeOH) 564 nm; m/z (Maldi): 560.

6.86,7,9,10-Tetrahydro-2-aminomethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium

A solution of6,7,9,10-tetrahydro-2-(N-formyl)aminomethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(2 mg) in conc. HCl: methanol [1:12] (5 ml) was stirred for 12 hours.The reaction solvent was removed in vacuo and the product purified byHPLC on a Phenomenex Jupiter C18, 10 μm column using 0-100% gradientelution of water/acetonitrile (containing 0.1% TFA) over 30 minutes at 4ml/min. The product was obtained as a pink solid (1.9 mg, 50%);λ_(max)(MeOH) 560 nm; m/z (Maldi): 532.

EXAMPLE 76,7,9,10-Tetrahydro-2-hydroxyethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VII)

7.1 5-Hydroxyethyl-2,3,3-trimethylindolenine

To a stirred solution of 4-(2-hydroxy)ethyl-aniline (10 g, 73 mmol) in a3:2 water: conc. HCl (73 ml) solvent mixture at <0° C. was addeddropwise a cooled (<0° C.) solution of sodium nitrite (6 g, 73 mmol) inwater (86 ml). The reaction mixture was then maintained at the reducedtemperature for a further 30 minutes. A saturated solution of sulphurdioxide (150 ml) was added and the reaction warmed to ambienttemperature over 1 hour, then warmed for a further hour at 70° C. Thereaction mixture was cooled rapidly and the solvent removed in vacuo.The yellow hydrazino intermediate product obtained was redissolved inacetic acid (120 ml) and potassium acetate (16 g, 163 mmol), andmethyl-isopropyl ketone (15.5 g, 180 mmol) added at ambient temperature.After 30 minutes the reaction mixture was warmed to 90° C. and stirredfor a further 2 hours. The reaction mixture was cooled and the solventremoved in vacuo. The product was dissolved in dichloromethane (100 ml)and washed with water (2×50 ml). The organic phase was dried over MgSO₄,filtered and concentrated in vacuo. The product was purified by HPLC ona Rainin Dynamax C18, 8 μm column using a 0-100% gradient elution ofwater/acetonitrile (containing 0.1% TFA) over 60 minutes at 20 ml/min.The product was obtained as a yellow oil (1.18 g, 8%); m/z (FAB⁺):204.1.

7.2 1-(3,3-Diethoxypropyl)-5-hydroxyethyl-2,3,3-trimethylindolenine

1-(3,3-Diethoxypropyl)-5-hydroxyethyl-2,3,3-trimethylindolenine wasprepared by reaction of 5-hydroxyethyl-2,3,3-trimethylindolenine (118mg, 0.072 mmol) with acrolein diethyl acetal (1.13 g, 8.68 mmol), aceticacid (100 μl) in acetonitrile (4 ml) by an analogous method to thatdescribed in Section 1.2. The product was purified by HPLC on aPhenomenex Jupiter C18, 10 μm column using a 0-100% gradient elution ofwater/acetonitrile (containing 0.1% TFA) over 30 minutes at 4 ml/min.The product was obtained as a pale brown oil (24 mg, 12%); m/z (Maldi):331.

7.35-Sulphonato-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenine

5-Sulphonato-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindoleninewas prepared by reaction of5-sulphonato-1-(3,3′diethoxypropyl)-2,3,3-trimethylindolenine (100 mg,0.27 mmol) [prepared as described in Section 3.2] withN,N′-diphenylformamidine (79 mg, 0.405 mmol) in acetic anhydride (20 ml)by an analogous method to that described in Section 3.3. The product wasnot purified, as decomposition was observed. The product was obtained asa yellow oil.

7.4 1-(3,3-Diethoxypropyl)-5-hydroxyethyl-indocarbocyanine

To a stirred solution of5-sulphonato-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenine(30 mg, 0.06 mmol) in 4.5:4.5:1 pyridine:acetic acid:acetic anhydride (5ml) at ambient temperature was added a solution of1-(3,3′-diethoxypropyl)-5-hydroxyethyl-2,3,3-trimethylindolenine (20 mg,0.06 mmol) in 4.5:4.5:1 pyridine:acetic acid:acetic anhydride (5 ml).The reaction mixture was warmed to 70° C. for 5 hours. The solution wascooled and the reaction solvent removed in vacuo. The product waspurified by HPLC on a Phenomenex Jupiter C18, 10 μm column using a0-100% gradient elution of water/acetonitrile (containing 0.1% TFA) over30 minutes at 4 ml/min. The product was obtained as a pink solid (8.6mg, 10%), λ_(max)(MeOH) 555 nm; m/z (Maldi):712.

7.56,7,9,10-Tetrahydro-2-hydroxyethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium

6,7,9,10-Tetrahydro-2-hydroxyethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-iumwas prepared by reaction of1,1-di-(3,3′diethoxypropyl)-5-hydroxyethyl-indocarbocyanine (4.3 mg,0.06 mmol) in chloroform (5 ml) and 50% sulphuric (1 ml) according tothe method described in Section 1.4. The product was purified by HPLC ona Phenomenex Jupiter C18, 10 μm column using a 0-100% gradient elutionof water/acetonitrile (containing 0.1% TFA) over 30 minutes at 4 ml/min.The product was obtained as a pink solid (2.8 mg, 90%), λmax 565 nm; m/z(Maldi): 547.

EXAMPLE 86,7,8,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-benzothiazolenine-indolenine-[3,2-a]-benzthiazolyl[3′2′-a]-pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VIII)

8.1 1-(3,3-Diethoxypropyl)-2-methylbenzothiazole

1-(3,3-Diethoxypropyl)-2-methyl-benzothiazole was prepared by reactionof 2-methylbenzothiazole (125 mg, 0.84 mmol) with acrolein diethylacetal (1.64 g, 12.6 mmol) and acetic acid (100 μl) in acetonitrile (4ml) by a method analogous to that described in Section 1.2. The productwas purified by HPLC on a Rainin Dynamax C18, 8 μm using a 0-100%gradient elution of water/acetonitrile (containing 0.1% TFA) over 60minutes at 20 ml/min. The product was obtained as a colourless oil (220mg, 95%); m/z (FAB⁺): 280.

8.25-Carboxymethyl-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenine,ethyl ester

5-Carboxymethyl-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenine,ethyl ester was prepared by reaction of5-carboxymethyl-1-(3,3-diethoxypropyl)-2,3,3-trimethylindoline, ethylester [prepared as described in Section 1.2] (1.16 g, 3.09 mmol) withN,N′-diphenylformamidine (908 mg, 4.63 mmol) in acetic anhydride (25 ml)by a method analogous to that described in Section 3.3. The product waspurified by HPLC on a Rainin Dynamax C18, 8 μm column using 10-100%gradient elution of water/acetonitrile (containing 0.1% TFA) over 60minutes at 20 ml/min. The product was obtained as a pale brown oil (634mg, 40%). m/z (FAB⁺): 521.

8.314-Carboxymethyl-1,1′-di(diethoxypropyl)-benzthiazolenine-indocarbocyanine,ethyl ester

To a stirred solution of5-carboxymethyl-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenine,ethyl ester (28 mg, 0.054 mmol) in 4.5:4.5:1 pyridine:acetic acid:aceticanhydride (5 ml) at ambient temperature was added a solution of1-(3,3-diethoxypropyl)-2-methyl-benzothiazole (15.1 mg, 0.054 mmol) in4.5:4.5:1 pyridine:acetic acid:acetic anhydride (5 ml). The reactionmixture was warmed to 70° C. for 5 hours. The solution was cooled andthe reaction solvent removed in vacuo. The product was purified by HPLCon a Phenomenex Jupiter C 8, 10 μm column using a 0-100% gradientelution of water/acetonitrile (containing 0.1% TFA) over 30 minutes at 4ml/min. The product was obtained as a pink solid (14.3 mg, 20%), λmax549 nm; m/z (FAB⁺): 665.3.

8.46,7,8,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-benzathiozolenine-indolenine-[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium

6,7,8,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-benzathiozolenine-indolenine-[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-iumwas prepared by reaction of (name)(5 mg, 0.0075 mmol) in chloroform (5ml) and 50% sulphuric acid (1 ml) by an analogous method to thatdescribed in section 1.4. The product was purified by HPLC on aPhenomenex Jupiter C18, 10 μm column using a 0-100% gradient elution ofwater/acetonitrile (containing 0.1% TFA) over 30 minutes at 4 ml/min.The product was obtained as a pink solid (3.2 mg, 90%); λmax 562 nm; m/z(Maldi): 471.

EXAMPLE 96,7,8,8a,9,10-Hexahydro-2,14-disulphonato-8-(4-carboxy-anilino)-16,16,18,18-tetramethyl-7aH-bis-indolinium[3,2-a;3′2′-a′]pyrido[3,2-c;5,6-c′]dipyridin-5-ium(Compound IX)

9.1 1,1-Di-(3,3-diethoxypropyl)-5,5′-disulphonato-indocarbocyanine

1,1-Di-(3,3-diethoxypropyl)-5,5′-disulphonato-indolcarbocyanine wasprepared by the reaction of1-diethoxypropyl-5-sulphonato-2,3,3-trimethylindolenine (25.4 mg, 0.069mmol) [prepared as described in Section 3.2] with triethyl orthoformate(40.7 mg, 0.275 mmol) in pyridine (5 ml) by an analogous method to thatdescribed in Section 1.3. The product was purified by HPLC on a RaininDynamax C18, 8 μm column using a 0-100% gradient elution ofwater/acetonitrile over 60 minutes at 20 ml/min. The product wasobtained as a pink solid (6.3 mg, 12%); λmax (MeOH) 555 nm; m/z(Maldi):750.

9.26,7,8,8a,9,10-Hexahydro-2,14-disulphonato-8-(4-carboxy-anilino)-16,16,18,18-tetramethyl-7aH-bis-indolinium[3,2-a;3′2′-a′]pyrido[3,2-c;5,6-c′]dipyridin-5-ium

To a stirred solution of1,1-di-(3,3-diethoxypropyl)-5,5′-disulphonato-indolcarbocyanine (2 mg,0.0027 mmol) in anhydrous acetic acid (2 ml) was added 4-hydrazinophenylacetic acid (0.89 mg, 0.0054 mmol) and reaction was warmed to 100° C.After 10 minutes the solution was cooled and the reaction solventremoved in vacuo. The product was purified by HPLC on a PhenomenexJupiter C18, 10 μm column using a 0-100% gradient elution ofwater/acetonitrile (containing 0.1% TFA) over 30 minutes at 8 ml/min.The product was obtained as two diastereomeric compounds, both werepink/purple solids (0.04 mg, 2%, 0.06 mg, 3%). λmax (MeOH) 563 nm; m/z(FAB⁺): 717.21, (FAB⁺): 717.20.

EXAMPLE 106,7,9,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-quinolino-indolenium-[3,2-a,3′2′-a]-pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound

10.1 1-[2-(1,3-Dioxalan-2-yl)ethyl]-2-methyl-quinoline bromide

2-Methyl quinoline (1.6 g, 0.011 mol) and 2-(2-bromoethyl)-1,3-dioxolane(7.7 g, 0.043 mol) were heated together at 85° C. for 16 hrs. On coolingthe reaction mixture was diluted with diethyl ether and the resultantsolid filtered off, washed with ether and dried.1-[2-(1,3-dioxalan-2-yl)ethyl]-2-methyl-quinoline bromide was obtainedas a brown solid (0.98 g, 27%). m/z (FAB⁺) 244.

10.25-Carboxymethyl-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenineethyl ester

5-Carboxymethyl-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenineethyl ester was prepared by reaction of5-carboxymethyl-1-(3,3-diethoxypropyl)-2,3,3-trimethylindoline ethylester [prepared as described in section 1.2] (1.16 g, 3.09 mmol) withN,N′-diphenylformamidine (908 mg, 4.63 mmol) in acetic anhydride (25 ml)by a method analogous to that described in Section 3.3. The product waspurified by HPLC on a Rainin Dynamax C18 column using 10-100% gradientelution of water/acetonitrile (containing 0.1% TFA) over 60 minutes at20 ml/min. The product was obtained as a pale brown oil (634 mg, 40%).m/z (FAB⁺): 521

10.314-Carboxymethyl-1-[2-(1,3-dioxalan-2-yl)ethyl]-1′-(3,3-diethoxypropyl)-quinolino-indocarbocyanine,ethyl ester

To a stirred solution of5-carboxymethyl-1-(3,3-diethoxypropyl)-2-(2-N-acetyl-N-phenylamino)ethenyl-2,3,3-trimethylindolenineethyl ester (16.1 mg, 0.031 mmol) in ethanol (0.5 ml) at ambienttemperature was added a solution of1-[2-(1,3-dioxalan-2-yl)ethyl]-2-methyl-quinoline-bromide (10 mg, 0.031mmol) in ethanol (0.5 ml) and triethylamine (125 μl, 0.9 mmol). After 1hour the reaction solvent was removed in vacuo and the product purifiedby HPLC on a Phenomenex Jupiter C18, 10 mm column using a 0-100%gradient elution of water/acetonitrile (containing 0.1% TFA) over 30minutes at 4 ml/min. The product was obtained as a purple solid (10 mg,51%), λmax 567 nm; m/z (FAB⁺): 629.

10.46,7,9,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-quinolino-indolenine-[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium

6,7,9,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-quinoline-indolenine-[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-iumwas prepared by reaction of(14-carboxymethyl-1-[2-(1,3-dioxalan-2-yl)ethyl]-1′-(3,3-diethoxypropyl)-quinolino-indocarbocyanine,ethyl ester (5 mg, 0.0079 mmol) in chloroform (2 ml) and 50% sulphuricacid (0.4 ml) by an analogous method to that described in section 1.4.The product was purified by HPLC on a Phenomenex Jupiter C18, 10 mmcolumn using a 0-100% gradient elution of water/acetonitrile (containing0.1% TFA) over 30 minutes at 4 ml/min. The product was obtained as apurple solid (1.6 mg, 44%); λmax 584 nm; m/z (FAB⁺):465.

EXAMPLE 11 Preparation of Rigid Cy-3-Cy-5 Conjugate (Compound (X)

To a mixture of O—(N-succinimidyl-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate (1 mg, 0.0024 mmol), and N,N′-diisopropylethylamine(0.68 mg, 0.007 mmol) in dimethyl sulphoxide (100 μl) at ambienttemperature was added6,7,9,10-tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(1 mg, 0.0018 mmol). After 1 hour diisopropylethylamine (0.23 mg, 0.0018mmol) and a solution of5-aminomethyl-5′-sulphonato-1-methyl-1′-ethylindodicarbocyanine (0.9 mg,0.0018 mmol) in dimethyl sulphoxide (100 μl) was added at ambienttemperature. After a further 48 hours the product was purified by HPLCon a Phenomenex Jupiter C18, 10 μm column using a 0-100% gradientelution of water/acetonitrile (containing 0.1% TFA) over 30 minutes at 4ml/min. The product was obtained as a blue solid; λ_(abs) (MeOH) 561 nmand λ_(em) 647 nm; m/z (FAB⁺): 1050.

EXAMPLE 12 Protein:Peptide Polarization Binding Assay 12.1 Synthesis ofLabelled Peptide Ligand

A peptide of sequence E-pY-I-N-Q-S-V-P-K (E9K) was prepared by solidphase synthesis on an Applied Biosystems 431A peptide synthesizer usingstandard methods and materials. An excess of6,7,9,10-tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a:3′2′-a′]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound III), N-hydroxysuccinimide ester was coupled in DMSO in thepresence of diisopropylethylamine to the free N-terminus of theprotected peptide whilst still attached to the solid phase. Afterdeprotection for two hours, the crude labelled peptide was purified byreverse phase HPLC, using a gradient from water/0.1% TFA towater:acetonitrile (40:60)/0.1% TFA over 60 minutes.

12.2 Binding Assay

Various concentrations of Grb2 glutathione-5-transferase fusion proteinand E9K labelled with Compound III in 20 mM MOPS pH7.4/10 mM DTT/005%Tween 20 were incubated in a final volume of 150 μl in black 96-wellmicroplates (Dynatech) for 60 minutes. Non-specific binding was definedusing 100M unlabelled peptide. Polarization values were read on aFluorolite FPM2™ plate reader (Jolley Research and Consulting Inc.)using a 530DF30 filter for excitation and 590DF45 filter for emission.The results are shown in FIG. 6 and indicate the specific binding (asdetermined by change in polarization) of the Compound III-labelledpeptide with the Grb2 protein.

EXAMPLE 13 Nucleic Acid FRET Hybridization Assay 13.1 Probe Preparations

Unlabelled target oligonucleotide (5′ TAC CCA GAC GAG CAA-biotin 3′) andcomplementary unlabelled probe oligonucleotide (5′ TTG CTC GTC TGG GTA3′) were synthesised on an Applied Biosystems 391 DNA synthesiser usingstandard methods and materials. The oligonucleotides were deprotectedfor 17 hours at 40° C. and purified by reverse phase HPLC using a C18column and a 40% TEM/acetonitrile gradient. The desired peaks werecollected, freeze dried and the samples were resuspended in sterile H₂O.

A second set of target and probe oligonucleotides were synthesised asdescribed, but an amino group was added to the 5′ terminal (5′ C7amino-modifier TAC CCA GAC GAG CAA-biotin 3′ and 5′ C7 amino modifierTTG CTC GTC TGG GTA 3′).

Amino modified target and probe oligonucleotides were incubated with a10-fold molar excess of6,7,9,10-tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a:3′2′-a′]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound III), N-hydroxysuccinimide ester and Cy5 NHS-ester dye(Amersham Pharmacia Biotech) respectively, in 0.1M sodium bicarbonatebuffer (pH9), overnight at 22° C. The following morning, theoligonucleotides were ethanol precipitated and the resulting pelletswere resuspended in H₂O. Labelled oligonucleotides were purified byreverse phase HPLC using a C18 column and a 60% TEAA/acetonitrilegradient and the desired peaks collected and freeze dried. Residues wereresuspended into H₂O and concentration of recovered material wasdetermined.

13.2 Binding Assay

Wells of a black, streptavidin coated 96-well plate were coated witheither unlabelled or Compound III-labelled target oligonucleotides (20pmol/well diluted in 100 μl PBS/1 MgCl₂) for 120 minutes at ambienttemperature. Any unbound material was removed by washing wellsvigorously with assay buffer (PBS/1 mM MgCl₂/0.1% BSA). Unlabelled orCy5-labelled probe oligonucleotides were diluted to 0.2 μmol/μl assaybuffer, and 100 μl was incubated with coated wells at ambienttemperatures for 120 minutes to allow probe hybridisation. Finally,wells were washed vigorously with PBS and fluorescence intensity wasmeasured on a fluorescence plate reader using a 560 nm excitation filterand a 670 nm emission filter (FIG. 7).

Wells coated with unlabelled target oligonucleotide and incubated witheither unlabelled or Cy5-labelled probe gave residual backgroundfluorescence signals. Similarly, wells coated with Compound III-labelledtarget oligonucleotide and incubated with unlabelled probe gave lowfluorescence signals. Wells coated with Compound III-labelled targetoligonucleotide and incubated with Cy5-labelled probe gave a strongfluorescence signal demonstrating that FRET can occur between CompoundIII and Cy5.

EXAMPLE 14 Protein:DNA Direct Intensity Binding Assay 14.1 Preparationof Reagents

All HPLC purified oligonucleotides were obtained from GenosysBiotechnologies Ltd. Equimolar amounts of a biotinylated coding strand(5′ Biotin-GATCTAGGGACTTT CCGCG (SEQ ID NO:5) 3′) and an unmodifiednon-coding strand (5′ ATCCCTGAAAGGCGCCTA 3′ (SEQ ID NO:6)) specific forNF-kB were incubated together in a boiling water bath for 3 minutes andallowed to anneal by cooling over 2 hours.

Anti-GST antibody (3 mg/ml, 1.5 mg supplied/vial (0.5 ml) from MolecularProbes) was dialysed against 1 litre of 0.15M sodium chloride for 4hours at room temperature and dialysis was continued overnight at 4° C.in a fresh solution of 0.15M sodium chloride. The following morning theantibody was dialysed against 1 litre of 0.1M sodium hydrogen carbonatefor a maximum of 4 hours.

A 1 mg/ml solution of6,7,9,10-tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a:3′2′-a′]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound II), N-hydroxysuccinimide ester in DMSO was added graduallywith stirring to the antibody at a ratio of 0.10 mg dye:0.33 mgantibody. The solution was mixed for a further 45 minutes at roomtemperature in the dark. Free dye was removed by dialysis against 1litre of 0.15M sodium chloride for 4 hours at room temperature andovernight at 4° C. against 1 litre of fresh 0.15M sodium chloride.Finally, the antibody was dialysed against 1 litre of 0.01M PBS/0.01%sodium azide for 4 hours at room temperature and then overnight at 4° C.against 1 litre of 0.01M PBS/0.01% sodium azide. [All dialyses wereperformed in the dark following labelling.]

14.2 Binding Assay

Biotin-labelled NF-kB-specific dsDNA (2.5 μmol, diluted in 0.01MMgCl₂)was added to each well (final volume, 100 μl) of a 96-wellstreptavidin coated microplate (Boehringer Mannheim) and incubated atroom temperature for 2 hours. Following washing with 0.01M phosphatebuffer (pH7.5) containing 0.05% Tween 20, 5 μmol/well of p65GST wasadded in 10 mM Hepes, 0.2 mM sodium acetate, 0.05% NP40, 1 mg/ml BSA and5 mM DTT (blanks contained no p65GST), in the presence or absence ofeither 200 μmol/well p65 (specific competitor) or casein (non-specificcompetitor). Both proteins were diluted in the Hepes buffer as above;final well volume was 100 μl. The plate was agitated at room temperaturefor 30 minutes and left to stand for a further 30 minutes. Followingwashing in PBS buffer as above, detection was achieved with 50 μmol/wellCompound III-labelled anti-GST Ab in Hepes buffer as above, 100 μl finalwell volume. Finally, the plate was washed with PBS buffer, as above and100 P analar water was added to each well. The plate was read atEx535/Em569 and Ex560/595 in the Biolumin 960 fluorescence microplatereader (Molecular Dynamics Inc.). The results are shown in FIG. 8.

Detection with Compound III-labelled anti-GST produced a good signal ofaround 10,000 rfu with a corresponding S/N ratio of between 101:1 (Ex560/Em595) and 123:1 (Ex535/Em569). Specificity was demonstrated using a40 fold molar excess of a specific competitor, p65 which reduced thetotal signal by approximately 90%. A non-specific competitor, caseinreduced the total signal by only 25%.

EXAMPLE 15 Protein:DNA FRET Binding Assay 15.1 Preparation of Reagents

All HPLC purified NF-kB-specific oligonucleotides were obtained fromGenosys Biotechnologies Ltd: a coding strand modified with a 5′ terminalprimary amine (5′ NH₂-GATCTAGGGACTTTCCGCG 3′ (SEQ ID NO:7)) and anunmodified non-coding strand (5′ ATCCCTGAAAGGCGCCTAG 3′ (SEQ ID NO:8)).A 10-fold molar excess of Cy-5-NHS ester dye (Amersham PharmaciaBiotech) was incubated with the coding strand, in 0.1M sodiumbicarbonate buffer (pH9), overnight at 22° C. The following morning theoligonucleotide was ethanol precipitated and then resuspended in water.The labelled coding strand was purified by reverse phase HPLC using aC18 column and a 60% TEAA/acetonitrile gradient. The peak containinglabelled oligonucleotide was freeze dried and resuspended in water.

NF-kB-specific double stranded (ds) DNA was generated by incubatingtogether equimolar amounts of the Cy-5 labelled coding strand (5′Cy5-GATCTAGG GACTTTCCGCG (SEQ ID NO:9) 3′) and the unmodified non-codingstrand (5′ ATCCCTGAAA GGCGCCTAG 3′ (SEQ ID NO:10)) in a boiling waterbath for 3 minutes and allowing to anneal by cooling over 2 hours.

NF-kB p65 protein (260 mg) was diluted to 1000 μl in 0.01M phosphatebuffered saline. A 20 fold molar excess of6,7,9,10-tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a:3′2′-a′]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound II), N-hydroxysuccinimide ester (as a 1 mg/ml solution inDMSO) was incubated with the protein at 22° C. with agitation for 2hours (in the dark). The labelled protein was dialysed against threechanges of 0.01M phosphate buffered saline/0.5M NaCl/3 mM EDTA/2 mM DTTat 4° C. (in the dark, 4 hours/fresh buffer).

15.2 Binding Assay

A black microtitre plate (Dynatech) was used for the FRET assay.Compound III-labelled p65 (20 μmol) was incubated in 10 mM Hepes, 0.2 mMsodium acetate, 0.05% NP40, 1 mg/ml BSA and 5 mM DTT with 10 μmol of Cy5labelled NF-kB-specific double stranded (ds) DNA in the presence orabsence of either 200 μmol/well p65 (specific competitor) or casein(non-specific competitor). Both proteins were diluted in the Hepesbuffer, to give a final well volume of 100 P. Wells containing CompoundIII-labelled p65 only were used as the blank. The plate was incubatedwith agitation for 30 minutes at 22° C. (in the dark) and left to standfor a further 30 minutes. The plate was read at Ex520/Em670 in theBiolumin 960 fluorescence microplate reader (Molecular Dynamics Inc.).The results are shown in FIG. 9.

A signal of 2000 rfu was obtained in the FRET assay with a correspondingS/N ratio of 4:1. Specificity was demonstrated using a 10 fold molarexcess of a specific competitor, p65, which reduced the total signal by40%. A non-specific competitor, casein had no effect on the totalsignal.

EXAMPLE 16 Receptor Ligand Binding Assay using Fluorescence Polarization16.1 Preparation of Reagents

A sample of6,7,9,10-tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a:3′2′-a′]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound II), N-hydroxysuccinimide ester (1 mg) was reacted with 1 mgof telenzepine amine congener* in dimethylsulphoxide in the presence of5% v/v triethylamine. The reaction was allowed to continue for 2 hoursat ambient temperature in the dark. The compound III-telenzepine productwas purified from the starting material by reverse phase HPLC using aC18 column and a 60% water/acetonitrile gradient in the presence of 0.1%trifluoracetic acid. The product peaks were collected, freeze dried inthe dark and resuspended in dimethyl sulphoxide. This was aliquoted andstored frozen at −20° C. in the dark. *Telenzepine amine congener wasprovided by Research Biochemicals International as part of the ChemicalSynthesis Programme of the National Institute of Mental Health, ContractN01 MH30003.

Chinese hamster ovary cells stably expressing the M₁ muscarinic receptor(CHO M₁ cells) were grown in HAMS F12 media (Sigma) with 10% foetalbovine serum (BRL); 2 mM glutamine; 50 IU/ml penicillin, streptomycin;125 μg/ml geneticin (Sigma), maintained at 37° C. with 5% CO₂ in ahumidified incubator. The cells were expanded into roller bottles,purged with 5% CO₂ and left in a roller bottle incubator at 37° C. for 4days.

The cells were harvested by scraping into cold phosphate buffered salinepH 7.3 (PBS tablets; Sigma) and pelleted by centrifugation at 1400 rcfat 4° C. in a MSE Mistral 3000i centrifuge. Cells were resuspended incold 5 mM MgCl₂, 50 mM Tris pH7.5 homogenisation buffer and left on icefor 20 minutes before cell lysis using the Parr cell disruptionapparatus (Parr Cat. No. 4639, 45 ml) using 900 psi of nitrogen. Anynon-disrupted cells were removed by centrifugation at 1400 rcf and thesupernatant was removed and further centrifuged at 1800 rpm in theBeckman J2-21M/E centrifuge for 20 minutes at 4° C. The resultingpellets were resuspended in cold homogenisation buffer and centrifugedat 18000 rpm as before. The cell membrane pellets were resuspended inapproximately 15 ml of homogenisation buffer, aliquoted and frozen inliquid nitrogen for storage at −70° C.

16.2 Binding Assay

Aliquots of CHO M₁ cell membrane (50 μg) was added to wells of a blackmicrotitre plate containing a range of dilutions of the M₁ muscarinicreceptor antagonists atropine and TAC (5000 nM-0.064 nM). The plate wasthen measured on the Fluorolite FPM-2™ where background polarizationvalues were determined (excitation filter 530 nm; emission filter 590nm). After addition of Compound III-telenzepine ligand (4 nM finalconcentration) the plate was sealed and incubated at 22° C. in the darkon a microtitre plate shaker. A final reading was taken after 80minutes. From the polarization data obtained, competition curves weregenerated for both atropine and TAC, using non-linear regression andone-site binding analysis (GraphPad Prism 2.0 data manipulationpackage).

The curves in FIG. 10 show specific polarization readings plottedagainst log₁₀ molar concentration unlabelled ligand. A specific signalof 190 mP was obtained and the IC₅₀ values determined from thisexperiment were 4.1 nM for atropine and 28 nM for the unlabelled TAC.

1. A method for covalently labelling a target material comprising thesteps of reacting together: i) a target material having at least onefunctional group selected from the group consisting of amino, hydroxyl,phosphoryl, carbonyl and sulphydryl groups; or having at least onereactive group that can covalently bond with said at least onefunctional group; and, ii) an amount of the compound (I) of formula:

(I) substituted by groups R²-R⁹, wherein groups R⁶, R⁷, R⁸ and R⁹ areattached to the atoms of the Z^(a) and Z^(b) ring structures; wherein,at least one of groups R² to R⁹ is a functional group selected from thegroup consisting of amino, hydroxyl, phosphoryl, carbonyl andsulphydryl; or wherein at least one of groups R² to R⁹ is a reactivegroup selected from succinimidyl ester, isothiocyanate, anhydride,haloacetamide, maleimide, sulphonyl halide, phosphoramidite, acidhalide, alkylimidate, hydrazide and carbodiimide that can covalentlybond with said at least one functional group; R¹ is selected fromhydrogen, aryl, heteroaryl, cyano, nitro, aldehyde, halogen, hydroxy,amino, quaternary amino, acetal, ketal, phosphoryl, sulphydryl, andalkyl groups optionally substituted by amino, C₁-C₄ alkyl-substitutedamino, quaternary amino, aldehyde, ketone, acetal, ketal, halo, cyano,aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate, amide andnitro; A is selected from O, S and NR¹¹ where R¹¹ is the substitutedamino radical:

where R′ is selected from hydrogen, a C₁₋₄ alkyl and aryl and R″ isselected from C₁₋₁₈ alkyl, aryl, heteroaryl, an acyl radical having from2-7 carbon atoms, and a thiocarbamoyl radical; X and Y may be the sameor different and are selected from bis-C₁-C₄ alkyl and C₄-C₅ spiro alkylsubstituted carbon, oxygen, sulphur, selenium, CH═CH, and N—W wherein Nis nitrogen and W is selected from hydrogen, a group —(CH₂)_(n)R¹² wheren is an integer from 1 to 26 and R¹² is selected from hydrogen, amino,aldehyde, acetal, ketal, halo, cyano, aryl, heteroaryl, hydroxyl,sulphonate, sulphate, carboxylate, substituted amino, quaternary amino,nitro, primary amide and substituted amide; Z^(a) and Z^(b) eachrepresent the atoms necessary to complete one, or two fused aromaticrings each ring having six carbon atoms; provided that when X and Y areother than carbon, at least one of R²-R⁹ represents -L-R¹⁰ or R¹⁰wherein R¹⁰ is said reactive group, or when X and Y are different andare selected from O and Se, at least one of R¹-R⁹ is other thanhydrogen, methyl, phenyl or naphthyl; wherein said reacting is performedfor a period of time sufficient to permit said at least one functionalor reactive group of said fluorescent compound to covalently bond tosaid at least one reactive or functional group of said target material.2. The method of claim 1 wherein R² to R⁹ are the same or different andare —R¹⁰ or -L-R¹⁰ where R¹⁰ is selected from hydrogen, halogen, amide,C₁-C₆ alkoxy, nitro, cyano, aryl, heteroaryl, sulphonate, quaternaryammonium, guanidinium, hydroxyl, phosphate, phosphonate, optionallysubstituted amino, azido, sulphydryl, carboxyl, carbonyl and reactivegroups selected from succinimidyl ester, isothiocyanate, anhydride,haloacetamide, maleimide, sulphonyl halide, phosphoramidite, acidhalide, alkylimidate, hydrazide and carbodiimide, and L is selected fromthe group consisting of a straight or branched C₁₋₂₀,alkyl chain, aC₂₋₂₀ monoether or polyether and a C₂₋₂₀ atom chain containing up tofour secondary amide linkages.
 3. The method of claim 1 wherein R¹ isselected from hydrogen, aryl, heteroaryl, cyano, halogen, alkyl groupsof twenty-six carbon atoms or less and —(CH₂)_(n)Q where 1<n<26 and Q isselected from amino, aldehyde, hydroxyl and groups reactive with amino,hydroxyl, aldehyde, phosphoryl, or sulphydryl groups and R², R³, R⁴ andR⁵ are hydrogen.
 4. The method of claim 1 wherein compound (I) isselected from: i)6,7,9,10-Tetrahydro-2,14-carboxymethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a;3′2′-a′]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound I); ii)8,9,11,12-Tetrahydro-3,17-disulphonato-20,20,22,22-tetramethyl-9aH,10aH-bisbenz[e]indolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-7-ium(Compound II); iii)6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound II); iv)6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium,glycinamide (Compound IV); v)6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium,N-(2-aminoethylcarboxamide) (Compound V); vi)6,7,9,10-Tetrahydro-2-(N-formyl)aminomethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VI); vii)6,7,9,10-Tetrahydro-2-hydroxyethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VII); viii)6,7,8,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-benzothiazolenine-indolenine-[3,2-a]-benzthiazolyl[3′2′-a]-pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VIII); ix)6,7,8,8a,9,10-Hexahydro-2,14-disulphonato-8-(4-carboxy-anilino)-16,16,18,18-tetramethyl-7aH-bis-indolinium[3,2-a;3′2′-a′]pyrido[3,2-c;5,6-c′]dipyridin-5-ium(Compound IX); x)6,7,9,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-quinolino-indolenium-[3,2-a,3′2′-a]-pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound X).
 5. The method of claim 1 wherein the compound (I) is6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-iumor its esters.
 6. The method of claim 5 wherein the ester is aN-hydroxysuccinimidyl ester.
 7. A method according to claim 1, whereinsaid target material is selected from antibody, lipid, protein, peptide,carbohydrate, nucleotides which contain or are derivatized to containone or more of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl,phosphate and thiophosphate groups, and oxy or deoxy polynucleic acidswhich contain or are derivatized to contain one or more of an amino,sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphategroups, microbial materials, drugs, toxins, particles, plastics or glasssurfaces and polymers.
 8. A target material selected from antibody,lipid, protein, peptide, carbohydrate, nucleotides which contain or arederivatized to contain one or more of an amino, sulphydryl, carbonyl,hydroxyl and carboxyl, phosphate and thiophosphate groups, and oxy ordeoxy polynucleic acids which contain or are derivatized to contain oneor more of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl,phosphate and thiophosphate groups, microbial materials, drugs, toxins,particles, plastics or glass surfaces and polymers herein said targetmaterial is covalently labelled with a compound (I) of formula:

(I) substituted by groups R²— R⁹, wherein groups R⁶, R⁷, R⁸ and R⁹ areattached to the atoms of the Z^(a) and Z^(b) ring structures; wherein,at least one of groups R² to R⁹ is a functional group selected from thegroup consisting of amino, hydroxyl, phosphoryl, carbonyl andsulphydryl; or wherein at least one of groups R² to R⁹ is a reactivegroup selected from succinimidyl ester, isothiocyanate, anhydride,haloacetamide, maleimide, sulphonyl halide, phosphoramidite, acidhalide, alkylimidate, hydrazide and carbodiimide that can covalentlybond with said at least one functional group; R¹ is selected fromhydrogen, aryl, heteroaryl, cyano, nitro, aldehyde, halogen, hydroxy,amino, quaternary amino, acetal, ketal, phosphoryl, sulphydryl, andalkyl groups optionally substituted by amino, C₁-C₄ alkyl-substitutedamino, quaternary amino, aldehyde, ketone, acetal, ketal, halo, cyano,aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate, amide andnitro; A is selected from O, S and NR¹¹ where R¹¹ is the substitutedamino radical:

where R′ is selected from hydrogen, a C₁₋₄ alkyl and aryl and R″ isselected from C₁₋₁₈ alkyl, aryl, heteroaryl, an acyl radical having from2-7 carbon atoms, and a thiocarbamoyl radical; X and Y may be the sameor different and are selected from bis-C₁-C₄ alkyl and C₄-C₅ spiro alkylsubstituted carbon, oxygen, sulphur, selenium, CH═CH, and N—W wherein Nis nitrogen and W is selected from hydrogen, a group —(CH₂)_(n)R¹² wheren is an integer from 1 to 26 and R¹² is selected from hydrogen, amino,aldehyde, acetal, ketal, halo, cyano, aryl, heteroaryl, hydroxyl,sulphonate, sulphate, carboxylate, substituted amino, quaternary amino,nitro, primary amide and substituted amide; Z^(a) and Z^(b) eachrepresent the atoms necessary to complete one, or two fused aromaticrings each ring having six carbon atoms; provided that when X and Y areother than carbon, at least one of R²— R⁹ represents -L-R¹⁰ or R¹⁰wherein R¹⁰ is said reactive group, or when X and Y are different andare selected from O and Se, at least one of R¹-R⁹ is other thanhydrogen, methyl, phenyl or naphthyl.
 9. The target material of claim 8wherein R² to R⁹ are the same or different and are —R¹⁰ or -L-R¹⁰ whereR¹⁰ is selected from hydrogen, halogen, amide, C₁-C₆ alkoxy, nitro,cyano, aryl, heteroaryl, sulphonate, quaternary ammonium, guanidinium,hydroxyl, phosphate, phosphonate, optionally substituted amino, azido,sulphydryl, carboxyl, carbonyl and reactive groups selected fromsuccinimidyl ester, isothiocyanate, anhydride, haloacetamide, maleimide,sulphonyl halide, phosphoramidite, acid halide, alkylimidate, hydrazideand carbodiimide, and L is selected from the group consisting of astraight or branched C₁₋₂₀ alkyl chain, a C₂₋₂₀ monoether or polyetherand a C₂₋₂₀ atom chain containing up to four secondary amide linkages.10. The target material of claim 8 wherein R¹ is selected from hydrogen,aryl, heteroaryl, cyano, halogen, alkyl groups of twenty-six carbonatoms or less and —(CH₂)_(n)Q where 1<n<26 and Q is selected from amino,aldehyde, hydroxyl and groups reactive with amino, hydroxyl, aldehyde,phosphoryl, or sulphydryl groups and R², R³, R⁴ and R⁵ are hydrogen. 11.The target material of claim 8 wherein compound (I) is selected from: i)6,7,9,10-Tetrahydro-2,14-carboxymethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a;3′2′-a′]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound I); ii)8,9,11,12-Tetrahydro-3,17-disulphonato-20,20,22,22-tetramethyl-9aH,10aH-bisbenz[e]indolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-7-ium(Compound II); iii)6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound III); iv)6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium,glycinamide (Compound IV); v)6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium,N-(2-aminoethylcarboxamide) (Compound V); vi)6,7,9,10-Tetrahydro-2-(N-formyl)aminomethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VI); vii)6,7,9,10-Tetrahydro-2-hydroxyethyl-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VII); viii)6,7,8,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-benzothiazolenine-indolenine-[3,2-a]-benzthiazolyl[3′2′-a]-pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound VIII); ix)6,7,8,8a,9,10-Hexahydro-2,14-disulphonato-8-(4-carboxy-anilino)-16,16,18,18-tetramethyl-7aH-bis-indolinium[3,2-a;3′2′-a′]pyrido[3,2-c;5,6-c′]dipyridin-5-ium(Compound IX); x)6,7,9,10-Tetrahydro-14-carboxymethyl-16,16-dimethyl-7a-8a-quinolino-indolenium-[3,2-a,3′2′-a]-pyrano[3,2-c;5,6-c′]dipyridin-5-ium(Compound X).
 12. The target material of claim 8 wherein the compound(I) is6,7,9,10-Tetrahydro-2-carboxymethyl-14-sulphonato-16,16,18,18-tetramethyl-7aH,8aH-bisindolinium[3,2-a,3′2′-a]pyrano[3,2-c;5,6-c′]dipyridin-5-iumor its esters.
 13. The target material of claim 12 wherein the ester isa N-hydroxysuccinimidyl ester.
 14. A compound of the formula

wherein R is methyl or ethyl, R² to R⁹ are the same or different and are—R¹⁰ or -L-R¹⁰ where R¹⁰ is selected from hydrogen, halogen, amide,C₁-C₈ alkoxy, nitro, cyano, aryl, heteroaryl, sulphonate, quaternaryammonium, guanidinium, hydroxyl, phosphate, phosphonate, optionallysubstituted amino, azido, sulphydryl, carboxyl, carbonyl and reactivegroups selected from succinimidyl ester, isothiocyanate, anhydride,haloacetamide, maleimide, sulphonyl halide, phosphoramidite, acidhalide, alkylimidate, hydrazide and carbodiimide, and L is selected fromthe group consisting of a straight or branched C₁₋₂₀ alkyl chain, aC₂₋₂₀ monoether or polyether and a C₂₋₂₀ atom chain containing up tofour secondary amide linkages; R¹ is selected from hydrogen, aryl,heteroaryl, cyano, nitro, aldehyde, halogen, hydroxy, amino, quaternaryamino, acetal, ketal, phosphoryl, sulphydryl, and alkyl groupsoptionally substituted by amino, C₁-C₄ alkyl-substituted amino,quaternary amino, aldehyde, ketone, acetal, ketal, halo, cyano, aryl,heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate, amide andnitro; A is selected from O, S and NR¹¹ where R¹¹ is the substitutedamino radical:

where R′ is selected from hydrogen, a C₁₋₄ alkyl and aryl and R″ isselected from C₁₋₁₈ alkyl, aryl, heteroaryl, an acyl radical having from2-7 carbon atoms, and a thiocarbamoyl radical; X and Y may be the sameor different and are selected from bis-C₁-C₄ alkyl and C₄-C₅ spiro alkylsubstituted carbon, oxygen, sulphur, selenium, CH═CH, and N—W wherein Nis nitrogen and W is selected from hydrogen, a group —(CH₂)_(n)R¹² wheren is an integer from 1 to 26 and R¹² is selected from hydrogen, amino,aldehyde, acetal, ketal, halo, cyano, aryl, heteroaryl, hydroxyl,sulphonate, sulphate, carboxylate, substituted amino, quaternary amino,nitro, primary amide and substituted amide; Z^(a) and Z^(b) eachrepresent the atoms necessary to complete one, or two fused aromaticrings each ring having six carbon atoms; provided that when X and Y areother than carbon, at least one of R²-R⁹ represents -L-R¹⁰ or R¹⁰wherein R¹⁰ is said reactive group, or when X and Y are different andare selected from O and Se, at least one of R¹-R⁹ is other thanhydrogen, methyl, phenyl or naphthyl.